We are kindly thankful to you for your attention and comments to our
review “Structural analysis of lipopolysaccharides from
Gram-negative bacteria” published in Biochemistry(Moscow). We apology profoundly for our mistake in quoting data
of Prof. Dr. Giovanni Matera et al. [1] and Prof.
Dr. Ulrich Zähringer et al. [2] on review p.
390: “In experiments with human whole blood, LPS from B.
henselae whose pentaacyl lipid A contains an acyloxyacyl residue
16:0[3-O-(28:0(27-OH))] also did not induce the release of TNF-α
[105]” [3]. In fact,
these experiments were performed with LPS from Bartonella
quintana rather than from Bartonella henselae as we reported
[1-3].

At the time of publishing of the review, the structure of lipid A from
B. quintana endotoxin, whose biological activity is described in
the above-mentioned sentence, was not characterized. Only structures of
lipid A from endotoxins of B. henselae, a bacterium related to
B. quintana were known [2]. Structural
variants of lipid A from B. henselae may include rare
acyloxyacyl residues of different composition: 12:0[3-O(26:0(25-OH))],
12:0[3-O(28:0(27-OH))], and 16:0[3-O(26:0(25-OH))] [2]. The authors of this work revealed that the
endotoxins from B. henselae were 1000 times less active than
those from Salmonella enterica sv. Friedenau in inducing IL-8
synthesis by human embryonic kidney 293 cells (HEK 293) transfected
with CD14 and Toll-like receptors [2]. In the early
study by the group of M. C. Liberto and A. Foca using human whole blood
it was determined that the endotoxins from B. quintana did not
induce TNF-α synthesis [1]. Antagonistic
activity of LPS from B. quintana was elucidated in the work of
C. Popa with coworkers [4].

LPS molecules, which exhibit low endotoxic activity but retain the
ability to interact with agonist-specific receptors blocking
agonist-mediated cellular responses, have been referred as endotoxin
antagonists. For instance, Re-LPS from B. quintana do not cause
the stimulation of human mononuclear cells to production of
TNF-α, IL-1β, or IL-6 up to tested concentration 1 μg/ml.
In addition, the application of tenfold excess of antagonists, Re-LPS
from B. quintana, was sufficient for prevention of agonistically
highly active S-LPS Escherichia coli-driven mRNA synthesis of
the mentioned cytokines [4]. Moreover, TNF-α
and IL-6 release from human peripheral blood mononuclear cells
activated by SR-LPS from Veillonella parvula ATCC 10790 were
significantly inhibited by preliminary exposure of the cells to Re-LPS
from B. quintana [5]. LPS with the similar
antagonistic properties are produced by phototrophic bacteria such as
Rhodobacter sphaeroides and Rhodobacter capsulatus [6] as well as by marine bacterium Marinomonas
communis ATCC 27118T [7]. Unlike
S-LPS from E. coli O111:B4 triggering already TNF-α
production in human whole blood at their concentration 10 ng/ml, LPS
from Rb. sphaeroides even at higher concentrations (up to 10
μg/ml) do not elicit such effect [6]. The lipid
A from Rb. sphaeroides upon simultaneously addition with
agonistically active lipid A from Salmonella minnesota R595 (1
ng/ml) or with LPS from Helicobacter pylori (100 ng/ml) inhibits
IL-8 production by human monocytes at antagonist concentrations 100
ng/ml or 10 μg/ml, respectively [8]. The main
structural features of lipids A from Rb. sphaeroides and Rb.
capsulatus are the presence in their fatty acid residues of rare
3-oxo and unsaturated groups (table). By using lipid A synthetic
analogs, the role of cis or trans configurations of
double bond in the establishment of peculiar activity and antagonistic
property of Rb. sphaeroides lipid A was elucidated [6]. It was shown that the capacity of the synthetic
analogs to block TNF-α release in response to S-LPS E.
coli O111:B4 (10 ng/ml) decreased in the series:
2′-trans-lipid A > LPS Rb. sphaeroides ≥
2′-cis-lipid A > 2-cis-lipid A. Thus, the lipid
A analog from Rb. sphaeroides with unsaturated bond in
trans configuration possessed the most pronounced antagonistic
activity [6]. Further, the reduction of 3-oxo and
unsaturated groups in fatty acid residues (14:0(3-oxo) and
14:0[3-O(cisΔ714:1)]) of natural lipid A Rb.
sphaeroides did not increase its endotoxic activity [12].

Synthetic compounds Rhiz 1/1, Rhiz 1/2 and Rhiz 1/3—analogs of
lipid A from Rhizobium sin-1—provide additional examples
of lipid A antagonists [11]. They differ from each
other by acyloxyacyl residue at N-2′ position of a glucosamine
(GlcN) residue (table). The exposure of differentiated Mono Mac 6 cells
to these compounds up to their concentration 100 μg/ml (10% serum)
led to nearly undetectable TNF-α production. The ability of
Rhizobium sin-1 lipid A analogs to inhibit TNF-α release
induced by S-LPS E. coli O55:B5 (10 ng/ml) was assessed and
decrease in the sequence: LPS Rhizobium sin-1 > Rhiz 1/2 ≥
Rhiz 1/1 > Rhiz 1/3 [11, 13]. These data indicate that the antagonistic
activity of lipid A Rhizobium sin-1 is favored by the presence
of long chain 27-hydroxyoctacosanoic fatty acid residue. This
conclusion is supported by the experiments showing that the
substitution of 27-hydroxyoctacosanoic fatty acid residue by the
relative shorter tetradecanoic acid residue (C14) devoid of hydroxyl
group such in compound Rhiz 1/3 causes significant reduction of
antagonistic activity of the studied synthetic analog [11, 13, 14].

The importance of fatty acid residue extension in the maintenance of
lipids A antagonistic activity was also reported [9]. Thus, lipid A synthetic analog—tetraacylated
compound No. 6 comprised of 3-hydroxydecanoic fatty acid residues
only—does not prevent endotoxin-induced IL-6 production. However,
another tetraacylated synthetic compound No. 5, differing from compound
No. 6 by substitution of two 3-hydroxydecanoic fatty acid residues at
N-2′ and N-2 positions of GlcN II and GlcN I by
3-hydroxytetradecanoic acid residues (table), has pronounced
antagonistic activity [9].

The role of fatty acid distribution between two glucosamine residues in
the manifestation of antagonistic activity by lipids A had been
evaluated using tetraacylated synthetic analogs of lipid A from
Porphyromonas gingivalis—compound No. 3 and compound No. 4
(table). The latter compound is characterized by asymmetrical [3 +
1] fatty acid residue distribution, whereas the former by a
pseudo-symmetrical one [2 + 2]. Comparing the ability of these
compounds to inhibit TNF-α release in response to S-LPS E.
coli O55:B5 from differentiated Mono Mac 6 cells revealed that
compound No. 3 was the most potent antagonist in comparison to compound
No. 4 [10].

LPS from Francisella tularensis possessing tetraacylated lipid A
with three 3-hydroxyoctadecanoic (C18) fatty acid residues in
pseudo-symmetrical distribution have no either agonistic or
antagonistic activities [15].

Taking these data into consideration, it can be concluded that the
tendency of lipid A to exhibit antagonistic activity is dependent on
its composition, with the most pronounced impact of the length and
distribution of lipid A fatty acids between two glucosamine residues
[10].

In conclusion, we would like to express our gratitude to colleagues of
Dr. Giovanni Matera from Catanzaro University (Italy) for broadening
the data regarding opportunistic infections caused by B.
quintana and biological activity of LPS from this bacterium as well
as for the possibility to discuss the dependence of lipid A
antagonistic activity on its composition.